Expression of a mammalian protein in laboratory cell lines is the most common approach used to study its biological functions, including localization, trafficking, translocation and interaction with other cellular factors (Chen et al., Proc Natl Acad Sci USA 90:6508-6512, 1993; Lemas et al., J Biol Chem 269:18651-18655, 1994; Molloy et al., EMBO J 13:18-33, 1994; Quon et al., Proc Natl Acad Sci USA 91:5587-5591, 1994). This approach can also serve to produce laboratory or industrial scale quantities of recombinant proteins, for instance for structural studies or therapeutic purposes (Grisshanuner and Tate, Q Rev Biophys 28:315-422, 1995; Mather et al., Methods Mol Biol 62:369-382, 1997; Freimuth, Genet Eng 28:95-104, 2007). Although overexpression in bacterial cells is often used to produce proteins on a large scale, in many cases the expressed mammalian proteins, especially membrane proteins, either mis-fold or do not retain proper function due to the lack of necessary posttranslational modifications. Mammalian proteins can be sub-cloned into a mammalian promoter-driven expression vector and expressed in a commonly used laboratory cell line, such as Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK 293) cells, HeLa (cervical cancer) cells, or NIH 3T3 (mouse embryonic fibroblast) cells, for the aforementioned purposes.
Oftentimes, this strategy relies on the availability of antibodies to the target protein for detection and confirmation that it is being expressed. Thus, the intended studies are problematic for newly discovered proteins against which no antibodies have been generated, or when the effectiveness or the specificity of the antibody is in question. These difficulties are circumvented if the target protein is tagged with a short epitope tag to which an effective antibody is available. Epitope-tagging a protein also facilitates the purification of the target protein, as antibodies to the epitope tag can be immobilized to matrixes for affinity chromatography (Jarvik and Telmer, Annu Rev Genet 32:601-618, 1998; Fritze and Anderson, Methods Enzymol 327:3-16, 2000).
Expressing and tagging soluble mammalian proteins is relatively simple, as expression vectors with various epitope tags and multicloning sites are widely available, and the subcloning process is straightforward. In contrast, expressing and studying membrane proteins in laboratory cell lines is more technically challenging. First, effective antibodies specific for membrane proteins are often difficult to generate. Second, because the majority of membrane proteins possess a signal peptide at their N-terminus that directs co-translational translocation of membrane proteins into the endoplasmic reticulum (ER) for cell surface expression, and this short peptide is proteolytically cleaved within the ER, tagging at the N-terminus of a membrane protein involves insertion of the epitope tag between the signal peptide and the mature membrane protein. Tagging at the N-terminus is often preferred, especially for Type Ia membrane proteins, the class to which most eukaryotic membrane proteins with single membrane-spanning regions belong. Since this group of membrane proteins exposes their N-terminus on the exterior side of the plasma membrane, tagging at the N-terminus may avoid possible functional interferences of their C-terminal, cytosolic portion, which often serves as the signal domain. In addition, the exterior portion of membrane proteins is often glycosylated, which is required for full biological function. Therefore, N-terminal tagging can interfere with the post-translational modification of membrane proteins.
Vectors with signal peptides and epitope tags have been previously constructed and used in various studies (Guan et al., J Biol Chem 267:21995-21998, 1992; Kobilka, Anal Biochem 231:269-271, 1995; den Hertog and Hunter, EMBO J 15:3016-3027, 1996; Zhou et al., Mol Immunol 33:1127-1134, 1996). However, these vectors were tailored for the expression of individual membrane proteins. They therefore have limited cloning sites available for the adaptation of either different epitope tags, or a variety of membrane proteins. Some signal peptides also result in cytotoxicity that leads to either mutations or lower expression level of the membrane protein when it is expressed heterologously. Thus, a need exists to develop mammalian expression modules that can be adapted to subclone, tag, and express a variety of mammalian membrane proteins in common laboratory cell lines.